Increased vascular permeability is a prominent feature of the adult respiratory distress syndrome, ischemic coronary artery disease, and atherosclerosis. The applicant has shown that alpha-thrombin, an important coagulation protease, induces a reversible reorganization of endothelial cell microfilaments and conformational changes intimately reorganization of endothelial cell microfilaments and conformational changes intimately associated with an increased albumin clearance across endothelial monolayers. The biochemical events by which thrombin induces cellular contraction, however, are unclear. The proposed research will test the hypothesis that thrombin-induced endothelial contractile events are mediated by G protein-coupled phosphoinositide hydrolysis resulting in increased [Ca++]i and Ca++- activated protein kinase-induced caldesmon and myosin light chain phosphorylation. Thrombin-mediated phosphorylation of endothelial myosin (light and heavy chain) and the actin binding protein caldesmon, will be measured in human umbilical vein and bovine pulmonary endothelial cells by radioimmunoblotting and 2-dimensional SDS PAGE techniques. The intracellular distribution of actin, myosin, and caldesmon will be analyzed by immunofluorescent microscopy and functional assessment of in vitro endothelial cell permeability will utilize a novel system developed by the applicant. Specific Aim #1 will investigate the role of guanine nucleotide regulatory proteins (G proteins) in the reorganization of the endothelial cytoskeleton using G protein activators (fluoride, GTP gamma S) as well as G protein inhibitors (GDP beta S, pertussis and cholera toxins). Specific Aim #2 will examine the role of the phosphoinositol metabolites, inositol triphosphate and diacylglycerol, in the transduction of signals which alter the endothelial cytoskeleton. The regulation of [Ca++]i, a critical second messenger regulating important cellular processes including myosin phosphorylation, will be examined in Specific Aim #3. In Specific Aim #4 the Ca++-dependent and -independent protein phosphorylation of caldesmon and myosin by protein kinase C, cAMP-protein kinase, Ca++-calmodulin protein kinase II, and myosin light chain kinase will be examined. The identity of the relevant kinase-phosphorylation profiles, and phosphopeptide mapping techniques. These studies which investigate the regulation of endothelial actomyosin activation may further enhance understanding of the role of contractile proteins in control of endothelial cell barrier function.